Concentrations of chain-breaking antioxidants were studied in the first 6 postnatal weeks in 29 healthy preterm infants (gestational age 30-35 wk). Vitamin C, uric acid, and sulfhydryl groups declined, whereas vitamin E rose and bilirubin followed its typical biphasic postnatal course. The influence of these changes on the plasma peroxyl radical trapping capacity was assessed in vitro (TRAP assay). The trapping capacity decreased postnatally and this appeared to be related to the coincident fall in uric acid concentrations. Results did not differ between babies fed with only preterm formula (n = 12) and those fed predominantly with human milk (n = 6), except for higher bilirubin and TRAP values in the breast-fed infants. There are major postnatal changes in the concentrations of the plasma chain-breaking antioxidants and this may influence the susceptibility of the preterm baby to oxygen toxicity.
The ability of plasma from newborn babies (cord blood) and adults to inhibit iron-induced lipid peroxidation was compared. The caeruloplasmin and transferrin concentrations, and latent iron-binding capacity were lower in the babies (p less than 0.001). The plasma of many of the babies had no latent iron-binding capacity and contained non-protein-bound iron (measured by the bleomycin assay). The in vitro ability of plasma to inhibit iron-induced liposome peroxidation by either ferroxidase antioxidant activity (caeruloplasmin) or iron-binding antioxidant activity (transferrin) was measured. The antioxidant activity in both assays was decreased in the babies (p less than 0.001). The percentage inhibition of peroxidation in the iron-binding antioxidant assay correlated positively with the latent iron-binding capacity (p less than 0.001) and negatively with the presence of bleomycin-detectable iron (p less than 0.02) in the babies. This assay produced stimulation of peroxidation in 42% of the babies but none of the adults. The diminished capacity of cord blood plasma to prevent iron-induced lipid peroxidation may predispose the newborn baby to the toxic effects of oxygen.
A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow-injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h-' and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow-injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, intercept = 0.6, n = 2 16, r = 0.977). The main advantages of the proposed method are high sample throughput, high sensitivity, and good reproducibility.
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